columns
Acclaim Trinity P1 Column
Superior Pharmaceutical and Counterion Separations
The Acclaim Trinity P1 column is based on Nanopolymer Silica Hybrid technology.
Superior Performance for
Pharmaceutical Applications
The Acclaim® Trinity™ P1 is a
unique, high-efficiency, silica-based
column specifically designed for simultaneous separation of pharmaceutical
drug substances and counterions, as
well as mixtures of acidic, basic, and
neutral drugs.
Innovative nanopolymer silica
hybrid (NSH) technology results in unparalleled chromatographic performance
and maximum flexibility in adjusting
selectivity during method development.
Column Chemistry
The Acclaim Trinity P1 column
is based on NSH technology, which
consists of high-purity porous spherical
silica particles coated with charged
nanopolymer particles. The inner-pore
area of the silica bead is modified with
a covalently bonded organic layer
that provides both reversed-phase and
anion-exchange retention properties.
Outer surface is modified with cationexchange functionality. This chemistry
ensures spatial separation of the anionexchange and cation-exchange regions,
Passion. Power. Productivity.
1
and allows both retention mechanisms
to function simultaneously and be
controlled independently.
Chromatographic Features
The Acclaim Trinity P1 column
provides solutions for easy and straightforward method development because
selectivity can be optimized by adjusting
the mobile phase ionic strength, pH,
and organic solvent—independently
or concurrently.
NSH column technology provides
the Acclaim Trinity P1 column with the
following features:
• Multiple retention mechanisms
including reversed-phase, anionexchange, and cation-exchange
• Adjustable selectivity by changing
mobile phase ionic strength, electrolyte type, pH, and organic solvent
• Ideal selectivity for simultaneous
separation of basic, neutral, and
acidic analytes
• Retention of ionic and ionizable
analytes without using ion-pairing
reagents
Pharmaceuticals
The Acclaim Trinity P1 column
provides optimum selectivity for various
pharmaceutical counterions and drug
substances using volatile mobile phases
(e.g., ammonium acetate). Detection
methods include Corona® Charged
Aerosol Detection® (CAD®), Evaporative
Light Scattering Detection (ELSD),
Ultra-violet (UV), and Mass Spectroscopy (MS). Other HPLC mobile phases,
such as phosphate buffers, can also
be used depending on the specific
application. The Corona CAD detector
uses a unique and innovative detection
method to detect nonvolatile analytes,
and offers performance benefits unequaled
by refractive index (RI), UV, ELS and
chemiluminescence nitrogen (CLN)
detectors. These benefits include high
sensitivity (sub to single digit nanogram),
excellent injection-to-injection reproducibility, wide dynamic range, and gradient
compatibility. Therefore, the Corona
CAD detector is the preferred detector
for the Acclaim Trinity P1 column for
simultaneous determination of Active
Pharmaceutical Ingredients (API) and
their counterions.
Determinations of Pharmaceutical
Counterions
Salt formation is a critical step in
drug development because it provides
improved biopharmaceutical and
physicochemical properties, ease of
purification and handling. Consequently,
100
1
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: 60/15/25 v/v/v CH3CN/0.1 M NH4OAc, pH 5.2/
DI H2O
Temperature: 30 °C
Flow Rate:
0.6 mL/min
Inj. Volume: 5 µL
Inj. Amount: 5 ng
Detection:
Corona ultra or Sedex-85 ELS detector
Sample:
NaCl (1 ppm based on Na+) in DI H2O
Corona ultra
(Nebulizer – 35 °C; Gain – 100 pA; Filter – Off)
Na+
Cl-
mV
Sedex-85 ELS detector
(Evaporation – 45 °C; Gain – 10; Filter – Off)
Detector
Na+
(S/N)
Na+
(LOD)
Cl(S/N)
Corona ultra
71
0.2 ng
27
0.9 ng
Sedex-85
13
1.2 ng
2
> 11 ng
0
0
1
2
Minutes
3
Cl(LOD)
5
4
26935
Figure 1. Simultaneous separation of Na+ and CI- ions.
300
Column:
Dimensions:
Mobile Phase
Temperature:
Flow Rate:
Inj. Volume:
Detection:
3
1
mV
4
Peaks:
2
8
5 6
7
10
9
0
0
3
6
9
12
Minutes
15
Acclaim Trinity P1, 3 µm
3.0 × 100 mm
60/40 v/v CH3CN/20 mM (total) NH4OAc, pH 5
30 °C
0.5 mL/min
2 µL
Corona ultra (Gain = 100 pA; Filter = med;
Neb Temp = 30 °C)
(50 to 100 ppm)
1. Choline
2. Tromethamine
3. Sodium
4. Potassium
5. Meglumine
6. Mesylate
7. Nitrate
8. Chloride
9. Bromide
10. Iodide
26936
Figure 2. Simultaneous separation of pharmaceutical counterions (isocratic method).
approximately 50% of all drug molecules
used in medicinal therapy are administered
as salts.
In pharmaceutical formulations,
the most commonly used counterions
are sodium and chloride ions. While no
other reversed-phase column can retain
or separate them, the Acclaim Trinity
P1 column overcomes this challenge
by providing excellent separation and
peak shapes for both analytes (Figure 1).
The Corona detector exhibits superior,
sub-ppm sensitivity compared to ELS
detectors—5x better detection limits for
sodium ion and 13x better for chloride ion.
2
Figure 2 demonstrates that the
Acclaim Trinity P1 column provides
ideal selectivity for separating pharmaceutical counterions (including both
cations and anions) using acetonitrile/
ammonium acetate mobile phase and a
Corona ultra™ detector. Baseline separation
of five cations and five anions is achieved
within a single run. Note that column
selectivity is designed such that cations
elute before anions. This is the first and
only column available that separates
both cations and anions simply and
reliably. The excellent selectivity for
pharmaceutical counterions is also
illustrated in Figure 3, where 16
commonly used pharmaceutical counterions
(6 cations and 10 anions) are baseline resolved
on a 50 mm column.
Basic amino acids are often used as
pharmaceutical counterions or in pharmaceutical formulation. Figure 4 demonstrates
the baseline separation of three amino acids
(histidine, lysine, and arginine) and their
counterion anion chloride, on a Trinity P1
column within 4 min.
Column:
Dimensions:
Mobile Phase:
Gradient:
Time (min)
A
B
C
Temperature:
Flow Rate:
Inj. Volume:
Detection:
400
4
2
5
mV
3
6
1
Simultaneous Separation of API and
Counterions
In pharmaceutical development,
determinations of APIs and counterions
are two important assays. Due to the
charge and/or hydrophobicity differences,
APIs and couterions are usually analyzed
by different chromatographic methods
that require different separation columns
and/or different instrumentation platforms.
For example, reversed-phase liquid
chromatography (RPLC) is most commonly
used for analyzing APIs with intermediate
to higher hydrophobicity, but it often fails to
provide adequate retention for hydrophilic
counterions. Ion chromatography (IC)
provides a reliable, selective, and highly
sensitive solution for counterions. As
shown in Figures 5 and 6, simultaneous
separations of hydrophobic and hydrophilic
acidic drugs and respective counterions
(naproxen sodium salt and penicillin G
potassium salt) can be achieved with baseline resolution and excellent peak shapes on
a 50 mm Trinity P1 column.
13
9
7
8
10 11
-10
0
2
7
15
60
60
60
10
10
35
35
35
0
0
5
5
5
90
90
30 °C
0.5 mL/min
2 µL
Corona ultra (Gain = 100 pA; Filter = med;
Neb Temp = 30 °C)
(50 to 100 ppm)
1. Procaine
9. Chloride
2. Choline
10. Bromide
3. Tromethamine
11. Iodide
4. Sodium
12. Phosphate
5. Potassium
13. Malate
6. Meglumine
14. Tartrate
7. Mesylate
15. Citrate
8. Maleate
16. Sulfate
Peaks:
14
12
Acclaim Trinity P1, 3 µm
3.0 × 50 mm
A: CH3CN; B: DI H2O; C: 0.2 M NH4OAc, pH 4
16
15
0
0
3
6
Minutes
9
12
15
26937
Figure 3. Simultaneous separation of pharmaceutical counterions (gradient method).
300
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: A: CH3CN
B: D.I. H2O
C: 0.2 M NH4OAc, pH 4.0
Gradient:
Time (min)
–5 0 0.1 4
%A:
50 50 50 50
%B:
35 35 35 0
%C:
15 15 15 50
Temperature: 30 °C
Flow Rate:
0.6 mL/min
Inj. Volume: 1 µL
Detection:
Corona ultra (Gain = 100 pA;
Filter = med; Neb Temp = 25 °C)
Samples:
Histidine HCl, lysine HCl, arginine HCl,
0.1 mg/mL in 50% CH3CN
Peaks:
1. Histidine
2. Lysine
3. Chloride
4. Arginine
2
4
3
mV
1
t0
0
0
2
Minutes
1
3
4
26938
Figure 4. Separation of basic amino acids and chloride.
1000 1
3.0 × 50 mm
Temperature: 30 °C
2
mV
Acclaim Trinity P1, 3 µm
Dimensions:
Mobile Phase: 80/20 v/v CH3CN/20 mM (total) NH4OAc, pH 5
2.0 mL/min
1
Column:
Flow Rate:
t0
1
2.5 µL
Detection:
Corona ultra (Gain = 100 pA; filter = med;
Neb Temp = 30 °C)
Sample:
Na, Naproxen (0.2 mg/mL in mobile phase)
Peaks:
0.6 mL/min
2
0.6 and 2.0 mL/min
Inj. Volume:
1. Na +
2. Naproxen
t0
ONa
0
H3CO
0
1
2
Minutes
3
4
27109
Figure 5. Separation of hydrophobic acidic API and the counterion (naproxen sodium salt).
3
Similarly, the Trinity P1 column can
retain and separate both hydrophobic and
hydrophilic basic APIs (trimipramine and
dimethylbiguanide, respectively) with
corresponding counterions (maleate and
chloride) using simple isocratic methods
(Figures 7 and 8).
Drug Formulations
Figure 9 shows separation of
Aleve® Sinus and Headache formula,
which contains pseudoephedrine•HCl
salt and naproxen sodium salt as active
ingredients. Considering the charge
difference and the significant difference
in hydrophobicity, a gradient method
is justified. First, the Na+ ion elutes
with low buffer concentration and low
organic solvent followed by elution of
pseudoephedrine. The Cl- ion elutes
after simultaneously increasing both
buffer and organic solvent and high
buffer concentrations. Because pseudoephedrine cannot be observed by
CAD due to its volatility, UV and CAD
detectors are used in series to provide
for complementary detection.
Another example is the separation
of a mixture of basic and acidic drug
substances in an Advil Allergy and
Sinus medication that contains pseudoephedrine, trimipramine maleate,
and ibuprofen (Figure 10). Using a
gradient elution with acetonitrile and
ammonium acetate buffer, all analytes
of interest are well separated, baseline
resolved, and free of interferences in
< 3 min.
400
O
NH H
N
O
Column:
S
O
Dimensions: 3.0 x 50 mm
CH3
O- K+
Temperature: 30 °C
Mobile Phase: 60/40 CH3CN/20 mM (total) NH4OAc, pH 5.2
Flow Rate:
2
1
Acclaim Trinity P1, 3 µm
CH3
0.6 mL/min
Inj. Volume: 2.0 µL
mV
Detection:
Corona ultra (Gain = 100 pA; Filter = med;
Neb Temp = 30 °C)
Sample:
Penicillin G, Potassium Salt
Peaks:
1. K+
(0.2 mg/mL in mobile phase)
t0
2. Penicillin G
0
0
1
2
Minutes
3
4
26940
Figure 6. Separation of hydrophilic acidic API and the counterion (penicillin G potassium salt).
300
1
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: 30/70 v/v CH3CN/60 mM (total) NH4OAc, pH 5
Temperature: 30 °C
Flow Rate:
0.5 and 1.8 mL/min
Inj. Volume: 2 µL
Detection:
UV, 254 nm
Sample:
Trimipramine maleate (0.5 mg/mL in mobile phase)
Peaks:
1. Trimipramine
1.8 mL/min
t0
2
1
mAU
2. Maleate
0.5 mL/min
t0
N
CH3
CH2 CH CH2 N
CH3
CH3
2
0
0
1
2
Minutes
3
O
O
NO C CH CH C CH
4
26941
Figure 7. Separation of hydrophobic basic API and the counterion (trimipramine maleate).
400
NH
1
H2N
NH
• HCI
N CH3
N
H
CH
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 x 50 mm
Mobile Phase: 20/80 v/v CH3CN/40 mM NH4OAc, pH 5.2
Temperature: 30 °C
Flow Rate:
0.6 mL/min
Inj. Volume: 2.0 µL
mV
2
Detection:
Corona ultra (Gain = 100 pA; Filter = med;
Neb Temp = 30 °C)
Sample:
1,1-Dimethylbiguanide•HCl
(0.2 mg/mL in mobile phase)
Peaks:
1. 1,1-Dimethylbiguanide
2. Chloride
t0
0
0
1
2
Minutes
3
4
Figure 8. Separation of hydrophilic basic API and the counterion C4H11N5.
4
26942
800
Corona ultra
Voltage [mV]
t0
3
0
–100
100 2
–10
4
UV, 254 nm
2
0
1
2
Retention Time [min]
Reproducible Manufacturing
Each Acclaim Trinity P1 column
is manufactured to strict specifications
to ensure column-to-column reproducibility. Each column is individually
tested and shipped with a qualification
assurance report.
Sample Preparation:
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
1. Grind one tablet (~0.62 g) into
Mobile Phase: A: CH3CN
fine powder
B: DI H2O
2. Weigh 62 mg of above powder
C: 0.1 M NH4OAc, pH 5.2
in a 20 mL sample vial
Gradient:
3. Add 10 mL of CH3CN/H2O
Time (min)
–4 0
0.1 1 4
(v/v, 1/1) solution
A: 20 20 20 80 80
4. Sonicate the vial containing sample
B: 65 65 65 0 0
suspension at 40 °C for 30 min
C: 15 15 15 20 20
5. Filter the extract with 0.2 µm
Temperature: 30 °C
membrane filter
Flow Rate:
0.6 mL/min
6. Dilute 3x with DI H2O
Inj. Volume: 1 µL
+
Detection:
UV at 254 nm and a Corona Peaks: 1. Na
2. Pseudoephedrine
ultra detector in series
Corona ultra settings:
3. CIGain = 100 pA; Filter = med; Neb. Temp = 30 °C
4. Naproxen
4
1
Absorbance [mAU]
High-Throughput Analysis
The adjustable selectivity of the
Acclaim Trinity P1 column makes it
useful for high-throughput analysis.
Figure 5 shows the Na+ ion and naproxen
separated within 3 min (lower trace).
By tripling the flow rate, the analysis
time can be reduced to < 1 min (upper
trace). Similarly, for separation of
the basic drug trimipramine and its
counterion, maleate, increasing flow
rate can accelerate the analysis from
3.2 min to < 1 min (Figure 7).
3
4
26943
Figure 9. Separation of mixture of acidic and basic APIs and counterions: Aleve™ Sinus and
Headache (OTC).
600
CH3
N
N
H
OH
O
H
OH
OH
H
CI
mAU
CH3
O
H
N
4
CH3
CH3
t0
2
1
3
0
0
1
2
Minutes
3
Column:
Acclaim Trinity P1, 3 µm
Dimensions: 3.0 × 50 mm
Mobile Phase: A: CH3CN
O
B: DI H2O
C: 0.2 M NH4OAc, pH 4.1
OH
Gradient:
Time (min)
–4
0 0.1 1 4
A: 25 25 25 80 80
B: 65 65 65
0 0
C: 10 10 10 20 20
Temperature: 30 °C
Flow Rate:
1 mL/min
Inj. Volume:
2 µL
Detection:
UV at 254 nm
Peaks:
1. Pseudoephedrine
2. Chlorpheniramine
3. Maleate
4. Ibuprofen
*Background subtraction applied
4
26944
Figure 10. Separation of mixture of acidic and basic APIs and counterions: Advil Allergy
and Sinus (OTC).
5
SPECIFICATIONS
Column Chemistry:
SCX, WAX, and RP Mixed-Mode
Operating Pressure Limit:
4500 psi
Operating pH range (recommended):
2.5–7.0 (3.0 to 6.0)
Operating flow rate range (recommended):
0.30–1.60 (0.4 to 1.0) mL/min for 3.0 mm
i.d. formats
0.15–0.80 (0.2 to 0.5) mL/min for 2.1 mm
i.d. formats
ORDERING INFORMATION
In the U.S., call (800) 346-6390 or contact the Dionex Regional Office nearest you. Outside the U.S., order through your local Dionex office or distributor. Refer the following
part numbers:
Acclaim Trinity P1 Columns
Part Numbers
Acclaim Trinity P1, 3 µm 3.0 × 100 mm................................................................ 071387
Acclaim Trinity P1, 3 µm 3.0 × 50 mm.................................................................. 071388
Acclaim Trinity P1, 3 µm 2.1 × 100 mm................................................................ 071389
Acclaim Trinity P1, Guard, 3 µm 3.0 × 10 mm (2 ea.)........................................... 071390
Acclaim Trinity P1, Guard, 3 µm 2.1 × 10 mm (2 ea.)........................................... 071391
Acclaim SST Guard Cartridge Holder V-2............................................................. 069580
Acclaim SST Guard Holder Kit,V-2 (Holder V2 and Coupler)............................. 069707
Acclaim SST Guard Coupler (V-2)........................................................................ 074188
Aleve is a registered trademark of Bayer Corporation.
Advil is a registered trademark of Wyeth Corporation.
Trinity and ultra are trademarks, and Acclaim, CAD, and Charged Aerosol Detection are registered trademarks of Dionex Corporation.
Passion. Power. Productivity.
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6
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